IDENTIFICATION OF A 54-KDA MITOCHONDRIAL ACETAMINOPHEN-BINDING PROTEIN AS ALDEHYDE DEHYDROGENASE

The covalent binding of acetaminophen (APAP) to mitochondrial proteins has been postulated to alter the function of the organelle and contri bute to the development of the hepatotoxicity upon APAP overdose. To i dentify the arylated proteins CD-1 mice were administered 600 mg/kg AP AP and Western blots of mitochondrial proteins collected 4 hr after do sing were probed with anti-APAP antibodies, Five proteins of approxima tely 75, 60, 54, 44, and 33 kDa were detected on 1-D gels. Immunostain ing of the 54-kDa protein was most intense. Mitochondria were subseque ntly fractionated into inner and outer membrane, matrix, and intermemb rane space using digitonin, sonication, and differential centrifugatio n. The 54-kDa target was most highly enriched in the inner membrane fr action. On 2-D gels this 54-kDa band was resolved into three arylated proteins with pls of 6.4, 6.6, and 7.1. The pi 7.1 protein was excised from 55 2-D gels, and, after tryptic digestion, the two best-resolved peptides were sequenced and found to be 100% identical to mitochondri al aldehyde dehydrogenase, Coincident with APAP covalent binding the s pecific activity of the enzyme decreased; by the time of maximal coval ent binding at 4 hr after APAP, the activity was 60% of control. Since the enzyme is an abundant mitochondrial dehydrogenase, its decreased activity may contribute to the impaired mitochondrial function observe d after APAP administration. (C) 1996 Academic Press, Inc.